ABSTRACT Microbiological studies reveal a direct association between early-childhood caries (ECC) and the presence of Candida albicans, along with high levels of Streptococcus mutans in plaque-biofilms. Previous in vitro and in vivo studies demonstrated that C. albicans and S. mutans develop a symbiotic relationship, enhancing the severity of dental caries. This bacterial-fungal interaction is mediated by S. mutans exoenzymes termed glucosyltransferases (Gtfs). The Gtfs binds avidly to the fungal surface and produces exopolysaccharides (EPS) that promotes the development of cariogenic cross-kingdom biofilms. Our previous R03 supported (DE025728) studies demonstrated that N- and O-linked mannans on the C. albicans cell wall play key roles in this process. Mutant strains defective in mannans showed severely reduced GtfB binding (vs wild type), which in turn impaired EPS production and abrogated mixed-species biofilm formation in vivo, revealing potential antibioflm targets. Thus, we propose to further elucidate the mechanisms of GtfB binding/EPS production, and assess whether an enzymatic strategy targeting the ligand-binding function could prevent cariogenic biofilm development. We will use readily available ?- (and ?-) mannosidases for mannan degradation on Candida cell wall and glucanohydrolases for EPS digestion in situ. We hypothesize that the enzyme combination therapy will disrupt the GtfB binding sites on C. albicans surface and concomitantly digest the EPS produced by S. mutans Gtfs, thereby blocking cross-kingdom biofilm formation and preventing the onset of severe caries in vivo. To support our hypothesis, Aim 1 will characterize the Gtf binding-function mechanism using genetics (mutant strains) and biochemical (enzymatic) approaches in conjunction with spectroscopy-fluorescence and biophysical methods. Specifically, we will assess the impact of mannan-cleavage on Gtf binding/activity and EPS production. In parallel, we will assess the optimal amounts and combinations of enzymes to disrupt C. albicans-S. mutans interactions and biofilm formation. The efficacy of optimized dosages to biofilms will be evaluated in Aim 2. Then, we will assess the disruption of biofilm development and cariogenicity on tooth- enamel using our newly developed super-resolution confocal-surface topography system. Real-time dynamics of cross-kingdom interaction, biofilm formation, in situ pH, metabolic activity, development of enamel lesions, and biofilm detachment will be observed. In addition, we will test clinical isolates of S. mutans and C. albicans from ECC-patients. The most effective dosage/combination of enzymes will be evaluated in vivo. In Aim 3, we will determine antibiofilm and anticaries efficacy of the enzymatic therapy using a well-established rodent model of ECC. We will investigate the impact of this therapeutic approach in preventing the onset and severity of caries lesions. The influences on bacterial-fungal levels and plaque microbiome will be also assessed. Successful completion of these aims will lead to a non-microbiocidal and antimicrobial independent approach to reduce a prevalent and costly biofilm-induced oral disease that affect a vulnerable children population.